The DNA mismatch repair (MMR) system is essential for maintaining the integrity of mammalian genomes by removing misincorporated nucleotides that result from erroneous replication and by mediating a DNA damage response after exposure to genotoxic agents. Mutations in mammalian MMR genes result in increased spontaneous mutation rates and strong predisposition to cancer. Eukaryotic MMR is a complex system that requires the interaction of several MutS and MutL proteins for the initiation of the repair reaction. Subsequent to mismatch recognition, downstream events are activated that lead to the excision of misincorporated or damaged nucleotides and the signaling of DNA damage-induced cell cycle arrest and apoptosis. Our research program focuses on elucidating the functions of the individual MutS homologs (Msh) in mammalian MMR and on assessing their importance for the suppression of cancer. In the past funding period, we found that mutations impairing the ATPase activity of the Msh2-Msh6 complex (termed MutS1) cause DNA repair deficiency but do not impair the DNA damage response function of the complex, allowing us to effectively separate these two functions. Our studies further demonstrated that DNA damage signaling by MutS1 is important for the suppression of tumorigenesis in the initial stages of the process. In addition, we demonstrated that MMR missense mutations could result in more heterogeneous cancer phenotypes than those caused by complete loss of function mutations. Based on these results, we hypothesize that both DNA repair and DNA damage signaling by MutS1 contribute to the suppression and tissue specificity of MMR-dependent tumorigenesis. In this continuing application, we plan to investigate this hypothesis by analyzing the contribution of MutS1 to the repair of oxidative DNA damage and assessing the significance of this repair for tumor suppression in MutS1 mutant mice. We will also utilize novel conditional Msh2 mutant mouse models to study the significance of the DNA repair and damage response functions of MutS1 for intestinal tumorigenesis and chemotherapeutic treatment. Finally, we propose to establish an in vivo system to analyze MMR complex formation and identify novel proteins that interact with MutS1 and participate in mismatch excision and DNA damage response in mouse tissues. PUBLIC HEALTH RELEVANCE: The DNA mismatch repair system (MMR) is essential for maintaining the integrity of mammalian genomes and defects in MMR are the cause of the cancer syndrome hereditary nonpolyposis colorectal cancer (HNPCC) and a significant number of sporadic cancers in humans. We are studying mouse lines with mutations in key MMR genes to determine the impact on DNA repair functions, in vivo mutator phenotypes and the ability of MMR to suppress tumor formation. Our studies will provide a better understanding of the biological functions of this important genome maintenance system and of the molecular mechanisms that prevent tumor formation in mammals.